U.S. patent number 5,902,881 [Application Number 08/811,233] was granted by the patent office on 1999-05-11 for reagent useful for synthesizing sulfurized oligonucleotide analogs.
This patent grant is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Daniel C. Capaldi, Zacharia S. Cheruvallath, Douglas L. Cole, Vasulinga T. Ravikumar.
United States Patent |
5,902,881 |
Cheruvallath , et
al. |
May 11, 1999 |
Reagent useful for synthesizing sulfurized oligonucleotide
analogs
Abstract
The present invention is directed to a method of synthesizing
sulfurized oligonucleotide analogs by reacting an oligonucleotide
analog containing a phosphorous(III) linkage with a dithiocarbonic
acid diester polysulfide having the formula ##STR1## to produce a
sulfurized oligonucleotide analog. The diester polysulfide reagent
is useful in solution and solid phase oligonucleotide analog
synthesis.
Inventors: |
Cheruvallath; Zacharia S. (San
Diego, CA), Ravikumar; Vasulinga T. (Carlsbad, CA), Cole;
Douglas L. (San Diego, CA), Capaldi; Daniel C. (San
Diego, CA) |
Assignee: |
ISIS Pharmaceuticals, Inc.
(Carlsbad, CA)
|
Family
ID: |
25205965 |
Appl.
No.: |
08/811,233 |
Filed: |
March 3, 1997 |
Current U.S.
Class: |
536/25.3;
536/25.33; 536/25.34; 568/22 |
Current CPC
Class: |
C07H
21/00 (20130101); C07C 329/14 (20130101) |
Current International
Class: |
C07C
329/14 (20060101); C07C 329/00 (20060101); C07H
21/00 (20060101); C07H 021/00 () |
Field of
Search: |
;536/25.3,25.33,25.34
;568/22 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
8902521 |
|
May 1991 |
|
NL |
|
9116331 |
|
Oct 1991 |
|
WO |
|
WO 94/15946 |
|
Jul 1994 |
|
WO |
|
WO 95/04065 |
|
Feb 1995 |
|
WO |
|
WO 95/32980 |
|
Dec 1995 |
|
WO |
|
9609406 |
|
Mar 1996 |
|
WO |
|
WO 97/19092 |
|
May 1997 |
|
WO |
|
Other References
Carey et al., Advanced Organic Chemistry, 3rd Ed., Part A:
Structure and Mechanisms, Plenum Press, New York, NY, 1990, only
pp. 473-475 supplied. .
H.C.P.F. Roelen et al., "A Study on the Use of Phenylacetyl
Disulfide in the Solid-Phase Synthesis of Oligodeoxynucleoside
Phosphorothioates", Recl. Trav. Chim. Pays-Bas, vol. 110, pp.
325-331, (1991) (Jul./Aug.). .
Mitsuo Kodomari et al., "A Convenient Synthesis of BIS(ACYL)
Disulfides Using Phase-Transfer Catalysis", Synthesis, Aug. (1981),
pp. 637-638. .
P.C.J. Kamer et al., "An Efficient Approach Toward the Synthesis of
Phosphorothioate Diesters Via the Schonberg Reaction", Tetrahedron
Letters, vol. 30, No. 48, pp. 6757-6760, (1989). .
Radhakrishnan P. Iyer et al., "A Novel Nucleoside Phosphoramidite
Synthon Derived From 1R,2S-Ephedrine", Tetrahedron: Asymmetry, vol.
6, No. 5, pp. 1051-1054, (1995). .
Kazunobu Miura et al., "Blockwise Mechanical Synthesis of
Oligonucleotides by the Phosphoramidite Method", Chem. Pharm. Bull,
vol. 35, No. 2, pp. 833-836, (1987). .
Willi Bannwarth, "Synthesis of Oligodeoxynucleotides by the
Phosphite-Triester Method Using Dimer Units and Different
Phosphorous-Protecting Groups", Helvetica Chimica Acta, vol. 68,
pp. 1907-1913, (1985) (Jul. 22). .
G. Kumar et al., "Improvements in Oligodeoxyribonucleotide
Synthesis: Methyl N,N-Dialkylphosphoramidite Dimer Units for Solid
Support Phosphite", J. Org. Chem., vol. 49, pp. 4905-4912, (1984).
.
Radhakrishnan P. Iyer et al., "Nucleoside Oxazaphosholidines as
Novel Synthons in Oligonucleotide Synthesis", J. Org. Chem., vol.
60, No. 17, pp. 5388-5389, (1995). .
Zhang et al. (I), "Synthesis and Properties of Novel Thiono
Triester Modified Antisense Oligodeoxynucleotide
Phosphorothioates," Bioorganic & Medicinal Chem. Letters,
5(15), 1735-1740 (Aug. 3, 1995). .
Zhang et al. (II), "Thiono Triester Modified Antisense
Oligonucleotides for Inhibition of Human Cytomegalovirus in Vitro,"
Bioorganic & Medicinal Chem. Letters, 6(16), 1911-1916 (Aug.
20, 1996). .
Bokarev et al., "Synthesis of Bis(Alkyl Xanthyl) Trisulfides," Izv.
Akad. Nauk SSSR, Ser Khim., 1964(12), 2175-2182; Chem. Abstr.,
62(7), Abstr. No. 7631d (Mar. 29, 1965); only Abstract supplied.
.
Scholl et al., "Novel Symmetrical and Mixed Carbamoyl and Amino
Polysulfanes by Reactions of (Alkoxydichloromethyl)polysulfuranyl
Substrates with N-Methylaniline," J. Organic Chem., 51(10),
1866-1881 (1986). .
Barany et al., "A General Strategy for Elaboration of the
Dithiocarbonyl Functionality, --(C.dbd.O)SS--: Application to the
Synthesis of Bischlorocarbonyl)disulfane and Related Derivatives of
Thiocarbonic Acids," J. Organic Chem., 48(24), 4750-4761 (Dec. 2,
1983)..
|
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Crane; L. Eric
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, P.C.
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. A method, comprising:
reacting an oligonucleotide analog comprising at least one
phosphorus(III) linkage with a thiodicarbonic acid diester
polysulfide having the formula: ##STR14## to produce a sulfurized
oligonucleotide analog, wherein each R is independently selected
from the group consisting of C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms; and
n is 2, 3 or 4.
2. The method of claim 1, wherein said phosphorus(III) linkage is a
phosphite triester or a thiophosphite triester.
3. The method of claim 1, wherein each R is independently selected
from the group consisting of C.sub.2 -C.sub.8 alkyl, substituted
C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14 aryl and substituted
C.sub.6 -C.sub.14 aryl; and
n is 2.
4. The method of claim 1, wherein said oligonucleotide analog
comprising at least one phosphorus(III) linkage has the formula:
##STR15## wherein each Pg is independently a group labile to a base
and/or a nucleophile or an allyl group;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or a protected heterocyclic
base;
each X is independently selected from the group consisting of
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6
-C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1
-C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1
-C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1
-C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
-C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8
-alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group;
each Y is independently O or S;
each Z is independently O or S;
L is a group labile to a nucleophile and/or a base;
S.sup.p is a solid support; and
m is 0 or a positive integer.
5. The method of claim 4, wherein each R is independently selected
from the group consisting of C.sub.2 -C.sub.8 alkyl, substituted
C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14 aryl and substituted
C.sub.6 -C.sub.14 aryl;
n is 2; and
m is an integer between 0 and 198.
6. The method of claim 5, wherein
each Pg is independently selected from the group consisting of
.beta.-cyanoethyl, 4-cyano-2-butenyl, 2-diphenylmethylsilyl and a
2-N-amidoethyl group having the formula R.sup.1 CONR.sup.2
CHR.sup.3 CHR.sup.4 --;
each R.sup.1 is independently selected from the group consisting of
C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms;
each R.sup.2, R.sup.3 and R.sup.4 is independently selected from
the group consisting of hydrogen, C.sub.1 -C.sub.8 alkyl,
substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14
aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
substituted C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, or
R.sup.3 and R.sup.4 together with the carbon atoms they are bonded
to form a C.sub.3 -C.sub.8 cycloalkyl group, a substituted C.sub.3
-C.sub.8 cycloalkyl group, a C.sub.2 -C.sub.8 heterocycloalkyl
group containing up to three heteroatoms or a substituted C.sub.2
-C.sub.8 heterocycloalkyl group containing up to three
heteroatoms;
R.sup.6 is selected from the group consisting of
4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl,
phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; and
each B is independently selected from the group consisting of
adenine, guanine, cytosine, thymine, uracil, 2-aminopurine, inosine
and 5-methylcytosine, where the exocyclic amino group of each base
is protected with an acyl group.
7. The method of claim 1, wherein said oligonucleotide analog
comprising at least one phosphorus(III) linkage is a dinucleotide
analog having the formula: ##STR16## wherein Pg is a group labile
to a base and/or a nucleophile;
R.sup.7 is a group labile to a base and/or a nucleophile;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or a protected heterocyclic
base;
each X is independently selected from the group consisting of
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6
-C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1
-C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1
-C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1
-C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
-C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8
-alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group; and
Y is O or S.
8. The method of claim 7, wherein each R is independently selected
from the group consisting of C.sub.2 -C.sub.8 alkyl, substituted
C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14 aryl and substituted
C.sub.6 -C.sub.14 aryl; and
n is 2.
9. The method of claim 8, wherein
Pg is selected from the group consisting of .beta.-cyanoethyl,
4-cyano-2-butenyl, 2-diphenylmethylsilyl and a 2-N-amidoethyl group
having the formula R.sup.1 CONR.sup.2 CHR.sup.3 CHR.sup.4 --;
R.sup.1 is selected from the group consisting of C.sub.1 -C.sub.8
alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14
aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
substituted C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms;
R.sup.2, R.sup.3 and R.sup.4 are independently selected from the
group consisting of hydrogen, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, or
R.sup.3 and R.sup.4 together with the carbon atoms they are bonded
to form a C.sub.3 -C.sub.8 cycloalkyl group, a substituted C.sub.3
-C.sub.8 cycloalkyl group, a C.sub.2 -C.sub.8 heterocycloalkyl
group containing up to three heteroatoms or a substituted C.sub.2
-C.sub.8 heterocycloalkyl group containing up to three
heteroatoms;
R.sup.6 is selected from the group consisting of
4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl,
phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; and
each B is independently selected from the group consisting of
adenine, guanine, cytosine, thymine, uracil, 2-aminopurine, inosine
and 5-methylcytosine, where the exocyclic amino group of each base
is protected with an acyl group; and
R.sup.7 is a C.sub.2 -C.sub.10 acyl group.
10. A method, comprising:
(a) providing a nucleoside analog having a blocked hydroxyl
group;
(b) deblocking said blocked hydroxyl group to produce a free
hydroxyl group;
(c) reacting said free hydroxyl group with a protected nucleoside
analog phosphoramidite having a blocked hydroxyl group to produce
an oligonucleotide analog comprising a phosphorous(III) linkage and
a blocked hydroxyl group;
(d) reacting said phosphorous(III) linkage with a reagent selected
from the group consisting of an oxidizing agent and a
dithiocarbonic acid diester polysulfide having the formula:
##STR17## to produce an oxidized or sulfurized phosphorous(V)
linkage, where at least one linkage is a sulfurized phosphorous(V)
linkage; and
(e) repeating steps (b) through (d) at least once to produce a
sulfurized oligonucleotide analog,
wherein
each R is independently selected from the group consisting of
C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms; and
n is 2, 3, or 4.
11. The method of claim 10, wherein
each R is independently selected from the group consisting of
C.sub.2 -C.sub.8 alkyl, substituted C.sub.2 -C.sub.8 alkyl, C.sub.6
-C.sub.14 aryl and C.sub.6 -C.sub.14 substituted aryl; and
n is 2.
12. The method of claim 10, wherein said nucleoside analog is
attached to a solid support.
13. The method of claim 10, wherein said oligonucleotide analog
comprising a phosphorous(III) linkage and a blocked hydroxyl group
has the formula: ##STR18## wherein each Pg is independently a group
labile to a base and/or a nucleophile or an allyl group;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or a protected heterocyclic
base;
each X is selected from the group consisting of hydrogen, hydroxyl,
F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8
alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl,
substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl
containing up to three heteroatoms, substituted C.sub.3 -C.sub.11
hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18
aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 alkyl, substituted O--C.sub.1
-C.sub.8 alkyl, O--C.sub.2 -C.sub.8 heterocycloalkyl containing up
to three heteroatoms, substituted O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, O--C.sub.6
-C.sub.14 aryl, substituted O--C.sub.6 -C.sub.14 aryl, O--C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
O--C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
O--C.sub.7 -C.sub.18 aralkyl, substituted O--C.sub.7 -C.sub.18
aralkyl, O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to
three heteroatoms, substituted O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, O--C.sub.1
-C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl, O--C.sub.1 -C.sub.8
alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino, O-tri-C.sub.1 -C.sub.8
-alkyl silyl, substituted O-tri-C.sub.1 -C.sub.8 -alkyl silyl,
NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1 -C.sub.8).sub.2,
NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1 -C.sub.8).sub.2 alkenyl,
S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1 -C.sub.8 alkenyl, NH.sub.2,
N.sub.3, NH--C.sub.1 -C.sub.8 -alkyl-NH.sub.2, polyalkylamino and
an RNA cleaving group;
each Y is independently O or S;
each Z is independently O or S;
L is a group labile to a nucleophile and/or a base;
S.sup.p is a solid support; and
m is 0 or a positive integer.
14. The method of claim 13, wherein
each step (d) is reacting said phosphorous(III) linkage with said
dithiocarbonic acid diester polysulfide;
each R is independently selected from the group consisting of
C.sub.2 -C.sub.8 alkyl, substituted C.sub.2 -C.sub.8 alkyl, C.sub.6
-C.sub.14 aryl and substituted C.sub.6 -C.sub.14 aryl;
n is 2; and
m is an integer between 0 and 198.
15. The method of claim 14, wherein
each Pg is independently selected from the group consisting of
.beta.-cyanoethyl, 4-cyano-2-butenyl, 2-diphenylmethylsilyl and a
2-N-amidoethyl group having the formula R.sup.1 CONR.sup.2
CHR.sup.3 CHR.sup.4 --;
each R.sup.1 is independently selected from the group consisting of
C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms;
each R.sup.2, R.sup.3 and R.sup.4 is independently selected from
the group consisting of hydrogen, C.sub.1 -C.sub.8 alkyl,
substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14
aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
substituted C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, or
R.sup.3 and R.sup.4 together with the carbon atoms they are bonded
to form a C.sub.3 -C.sub.8 cycloalkyl group, a substituted C.sub.3
-C.sub.8 cycloalkyl group, a C.sub.2 -C.sub.8 heterocycloalkyl
group containing up to three heteroatoms or a substituted C.sub.2
-C.sub.8 heterocycloalkyl group containing up to three
heteroatoms;
R.sup.6 is selected from the group consisting of
4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl,
phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; and
each B is independently selected from the group consisting of
adenine, guanine, cytosine, thymine, uracil, 2-aminopurine, inosine
and 5-methylcytosine, where the exocyclic amino group of each base
is protected with an acyl group.
16. The method of claim 12, further comprising:
(f) removing said sulfurized oligonucleotide analog from said solid
support.
17. The method of claim 13, further comprising:
(f) removing said sulfurized oligonucleotide analog from said solid
support, wherein said sulfurized oligonucleotide analog removed
from said solid support has the formula: ##STR19##
18. The method of claim 14, further comprising: (f) removing said
sulfurized oligonucleotide analog from said solid support, wherein
said sulfurized oligonucleotide analog removed from said solid
support has the formula: ##STR20##
19. The method of claim 1, wherein the reacting step is conducted
at a temperature of 0 to 40.degree. C.
20. The method of claim 1, wherein the reacting step is conducted
at a temperature of 10 to 40.degree. C.
21. The method of claim 1, wherein the reacting step is conducted
for 30 seconds to 15 minutes.
22. The method of claim 1, wherein the reacting step is conducted
at a temperature of 0 to 40.degree. C. for 30 seconds to 15
minutes.
23. A composition, comprising: (a) an oligonucleotide analog
comprising at least one phosphorus(III) linkage; and
(b) an effective amount of a thiodicarbonic acid diester
polysulfide having the formula: ##STR21## for sulfurizing said at
least one phosphorus(III) linkage of the oligonucleotide analog,
wherein
each R is independently selected from the group consisting of
C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms and substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms; and
n is 2, 3 or 4.
24. The composition of claim 23, wherein n is 2.
25. The composition of claim 23, wherein said oligonucleotide
analog comprising at least one phosphorus(III) linkage has the
formula: ##STR22## wherein each Pg is independently a group labile
to a base and/or a nucleophile or an allyl group;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or a protected heterocyclic
base;
each X is independently selected from the group consisting of
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6
-C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1
-C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1
-C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1
-C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
-C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8
-alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group;
each Y is independently O or S;
each Z is independently O or S;
L is a group labile to a nucleophile and/or a base;
S.sup.p is a solid support; and
m is 0 or a positive integer.
26. The composition of claim 23, wherein said oligonucleotide
analog comprising at least one phosphorus(III) linkage is a
dinucleotide analog having the formula: ##STR23## wherein Pg is a
group labile to a base and/or a nucleophile;
R.sup.7 is a group labile to a base and/or a nucleophile;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or a protected heterocyclic
base;
each X is independently selected from the group consisting of
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6
-C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1
-C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1
-C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1
-C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8
-alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group; and
Y is O or S.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a method of synthesizing
sulfurized oligonucleotide analogs by reacting an oligonucleotide
analog containing a trivalent phosphorous linkage with a
dithiocarbonic acid diester polysulfide.
2. Discussion of the Background
It is well-known that most of the bodily states in mammals,
including most disease states, are effected by proteins. By acting
directly or through their enzymatic functions, proteins contribute
in major proportion to many diseases in animals and man.
Classical therapeutics has generally focused on interactions with
such proteins in an effort to moderate their disease causing or
disease potentiating functions. Recently, however, attempts have
been made to directly inhibit the production of proteins involved
in disease by interacting with the messenger RNA (mRNA) molecules
that direct their synthesis. These interactions have involved the
hybridization of complementary, or antisense, oligonucleotides or
oligonucleotides analogs to mRNA. Hybridization is the
sequence-specific hydrogen bonding of an oligonucleotide or
oligonucleotide analog to an mRNA sequence via Watson-Crick
hydrogen bond formation. Interfering with the production of
proteins involved in disease would provide maximum therapeutic
results with minimum side effects.
The pharmacological activity of antisense oligonucleotides and
oligonucleotide analogs depends on a number of factors that
influence the effective concentration of these agents at specific
intracellular targets. One important factor for oligonucleotides
and analogs thereof is their stability to nucleases. It is unlikely
that unmodified oligonucleotides containing phosphodiester linkages
will be useful therapeutic agents because they are rapidly degraded
by nucleases. Modified oligonucleotides which are nuclease
resistant are therefore greatly desired.
Phosphorothioate and phosphorodithioate oligonucleotide analogs
which have one or both of the non-bridging oxygens of the natural
phosphodiester linkage replaced with sulphur, respectively, are
especially promising antisense therapeutics. These oligonucleotide
analogs are highly resistant to nucleases, have the same charge as
natural phosphodiester-containing oligonucleotides, and are taken
up by cells in therapeutically effective amounts. See, for example,
Baracchini et al, U.S. Pat. No. 5,510,239; Ecker, U.S. Pat. No.
5,512,438; Bennett et al, U.S. Pat. No. 5,514,788; and Ecker et al,
U.S. Pat. No. 5,523,389.
Phosphorothioate and phosphorodithioate oligonucleotide analogs are
conveniently synthesized with automated DNA synthesizers using
hydrogen phosphonate chemistry which permits the phosphonate
backbone to be sulfurized in a single step after automated
synthesis. One drawback of this approach is that coupling yields
during chain synthesis are typically lower than those obtained
using phosphoramidite chemistry. The final yield of the desired
oligonucleotide analog is therefore too low due to the low
individual coupling yields.
Automated synthesis using phosphoramidite chemistry is a highly
desirable approach to the synthesis of these sulfurized
oligonucleotide analogs, with coupling yields typically greater
than 99%. However, the phosphorous(III)-containing phosphite
intermediates are unstable under the conditions of the
detritylation step of the synthesis cycle. Therefore, these
phosphorous(III) linkages must be sulfurized after each coupling
step.
A more recent method for the synthesis of oligonucleotide analogs
is the "blockmer" approach. In blockmer synthesis, an
oligonucleotide analog is made by the sequential coupling of short
protected oligomers or blocks, e.g., a dinucleotide, on a solid
support. This strategy offers several advantages over the
conventional synthetic approach which involves the sequential
coupling of monomeric nucleoside phosphoramidites. The number of
synthesis cycles required to prepare an oligonucleotide analog is
reduced, saving time and minimizing reagent consumption. The blocks
may be prepared on a large scale using inexpensive solution phase
synthesis techniques. In order to prepare sulfurized
oligonucleotide analogs by the blockmer method, a reagent for
sulfuring the phosphorous(III) linkages of the blocks on a large
scale is required. The blockmer approach is described in the
following references: Ravikumar et al, WO 95/32980; WO 94/15947;
Journal of Organic Chemistry 1984, 49, 4905-4912; Helevetica
Chimica Acta 1985, 68, 1907-1913; Chem. Pharm. Bull. 1987, 35,
833-836.
There are several reagents available for sulfurizing the phosphite
intermediates during automated oligonucleotide synthesis. All of
these reagents have drawbacks which limit their use for
synthesizing sulfurized oligonucleotide analogs.
Elemental sulfur, for example, has been used to sulfurize
phosphorous(III) linkages in solid phase oligonucleotide synthesis.
However, elemental sulphur is not suitable for use with automated
synthesizers because of its poor solubility in standard solvents
and slow sulfurization rate. In addition, carbon disulfide, the
preferred solvent for elemental sulphur, is highly volatile and has
a low flash point. See, U.S. Pat. No. 5,252,723 and U.S. Pat. No.
5,449,769.
The Beaucage reagent, 3H-1,2-benzodithiol-3-one, is a considerably
more efficient sulfurizing agent. However, this reagent
precipitates from solution and clogs the solvent and reagent
transfer lines of an automated DNA synthesizer. Also, the
by-product formed during the sulfurization reaction is a potent
oxidizing agent. This by-product can lead to side products, e.g.,
phosphodiesters, which are difficult to separate from the desired
sulfurized oligonucleotides. In addition, the preparation of this
reagent involves expensive and toxic materials, and is therefore
not amenable for large-scale synthesis of sulfurized
oligonucleotide analogs. See, U.S. Pat. No. 5,003,097.
Tetraethylthiuram disulfide is an inexpensive and chemically stable
sulfurization reagent. However, the sulfurization rate is slow and
therefore a significant molar excess of this reagent is required.
Even with an excess of this reagent, sulfurization yields are
unacceptably low. See, U.S. Pat. No. 5,166,387.
Phenylacetyl disulfide may be used to sulfurize phosphite
intermediates during automated oligonucleotide synthesis. However,
this reagent has not been reported to be useful for large-scale
synthesis of sulfurized oligonucleotide analogs. See, Recherches
Travaux Chimiques des Pays-Bas 1991, 110, 325-331; Tetrahedron
Letters 1989, 30, 6757-6760; Synthesis 1981, 637-638.
Accordingly, there remains a need in the art for methods and
reagents for synthesizing sulfurized oligonucleotide analogs which
overcome these problems.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of
synthesizing a sulfurized oligonucleotide using a reagent that
efficiently sulfurizes a phosphorous(III) linkage in an
oligonucleotide analog.
Another object of the present invention is to provide a method of
synthesizing a sulfurized oligonucleotide using a reagent that does
not require the use of solvents having a high volatility and flash
point.
Another object of the present invention is to provide a method of
synthesizing a sulfurized oligonucleotide using a reagent that is
highly soluble in organic solvents and does not precipitate from
solution.
Another object of the present invention is to provide a method of
synthesizing a sulfurized oligonucleotide using a reagent that is
useful in automated solid phase synthesis of oligonucleotide
analogs.
Another object of the present invention is to provide a method of
synthesizing a sulfurized oligonucleotide using a reagent that is
compatible with large-scale and small-scale synthesis using
solution phase methods.
Another object of the present invention is to provide a sulfurizing
reagent composition that may be used to sulfurize a
phosphorous(III) linkage of an oligonucleotide analog.
These objects and others may be accomplished with a method of
synthesizing a sulfurized oligonucleotide analog by reacting an
oligonucleotide analog containing a phosphorus(III) linkage capable
of being sulfurized with a thiodicarbonic acid diester polysulfide
having the formula: ##STR2## where each R is an inert side chain,
and n is 2, 3 or 4.
The above objects may also be accomplished with a method of
synthesizing a sulfurized oligonucleotide analog by:
(a) providing a nucleoside analog having a blocked hydroxyl
group;
(b) deblocking the blocked hydroxyl group to produce a free
hydroxyl group;
(c) reacting the free hydroxyl group with a protected nucleoside
analog phosphoramidite or a protected nucleoside analog
phosphorothioamidite having a blocked hydroxyl group to produce an
oligonucleotide analog containing a phosphorous(III) linkage and a
blocked hydroxyl group;
(d) reacting the phosphorous(III) linkage with a reagent selected
from the group consisting of an oxidizing agent and a
dithiocarbonic acid diester polysulfide to produce an oxidized or
sulfurized phosphorous(V) linkage;
(e) repeating steps (b) through (d) at least once to produce a
sulfurized oligonucleotide analog, wherein at least one step (d) in
the method is reacting the phosphorous(III) linkage with the
dithiocarbonic acid diester polysulfide of the present
invention.
The above objects may also be accomplished with a sulfurizing
reagent composition containing an effective amount of
thiodicarbonic acid diester polysulfide for sulfurizing a
phosphorous(III) linkage of an oligonucleotide analog and at least
one solvent.
DETAILED DESCRIPTION OF THE INVENTION
A more complete appreciation of the invention and many of the
attendant advantages thereof will be readily obtained as the same
becomes better understood by reference to the following detailed
description.
As used in the present invention, the term "oligonucleotide analog"
includes linear oligomers of natural or modified nucleosides linked
by phosphodiester bonds or analogs thereof ranging in size from two
monomeric units to several hundred monomeric units. Oligonucleotide
analogs include modifications of the heterocyclic base moiety
and/or the sugar portion of a component nucleotide. In particular,
the term includes non-natural oligomers containing phosphorus(III)
linkages which are amenable to sulfurization. Preferably, the
modifications do not inhibit the ability of an oligonucelotide
analog to bind to a target nucleic acid. The term "sulfurized
oligonucleotide analog" is an oligonucleotide analog containing at
least one analog of a phosphodiester linkage in which one or both
of the non-bridging oxygen atoms are replaced by sulfur. The term
"nucleoside analog" refers to a natural or modified nucleoside. In
particular, this term includes nucleosides that are modified at the
heterocyclic base and/or sugar to enhance hybridization to the
target nucleic acids. It is to be understood that the
stereochemical relationship between the sugar substituents in the
nucleoside and oligonucleotide analogs disclosed herein is
preferably the same as that of naturally-occurring DNA and RNA, see
G. M. Blackburn and M. J. Gait (eds.), Nucleic Acids in Chemistry
and Biology (ILR Press, 1990), Chapter 2, pp. 19-70.
The thiodicarbonic acid diester polysulfide of the present
invention preferably has the formula: ##STR3## where each R is
preferably an inert group. These groups preferably do not contain
any reactive moieties which could lead to side reactions or poor
yields in the sulfurization reaction. Preferably, each R is
independently C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8
alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl,
substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl
containing up to three heteroatoms, substituted C.sub.3 -C.sub.11
hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18
aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms or
substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to
three heteroatoms. The term "C.sub.1 -C.sub.8 alkyl" includes
linear, branched and cyclic alkyl groups. The term "substituted"
means that up to three hydrogen atoms in the group are substituted
with up to three halogen, nitro, cyano, C.sub.1 -C.sub.8 alkyl,
O--C.sub.1 -C.sub.8 alkyl, N--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
-C.sub.8 alkyl groups or combinations thereof. More preferably,
each R is independently C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14
aryl or substituted C.sub.6 -C.sub.14 aryl. Most preferably, each R
is independently ethyl or p-chlorophenyl. The dithiocarbonic acid
diester polysulfide may be a disulfide, trisulfide or tetrasulfide,
i.e., n is 2, 3 or 4, respectively. Preferably n is 2 or 3, and
more preferably, n is 2.
The thiodicarbonic acid diester polysulfide may be prepared by
oxidation of the corresponding thiodicarbonic acid or acid salt
with oxidizing agents, such as iodine or bromine. Methods of
synthesis and properties of these polysulfides are described in the
following references: Barany et al, Journal of Organic Chemistry,
1983, 48, 4750-4761; W. F. Zeise, J. Prakt. Chem. 1845, 36,
352-362; Losse et al., J. Prakt. Chem. 1961, 13, 260; S. R. Rao,
Xanthates and Related Compounds, (M. Decker, New York, 1971);
Bulmer et al, J. Chem. Soc. 1945, 674-677.
The thiodicarbonic acid diester polysulfide of the present
invention is an efficient reagent for sulfurizing phosphorous(III)
linkages in oligonucleotide analogs. The reagent does not
precipitate out of solution, even on prolonged storage. The reagent
may be used in solution phase synthesis and is particularly useful
in solid-phase synthesis of oligonucleotide analogs with automated
DNA synthesizers. The reagent is particularly useful in large-scale
synthesis, using either solution phase or solid phase
techniques.
When used to sulfurize phosphorus(III) linkages in oligonucleotide
analogs, the thiodicarbonic acid diester polysulfide is preferably
delivered to the oligonucleotide analog in a suitable organic
solvent, such as acetonitrile, pyridine, tetrahydrofuran,
dichloromethane, dichloroethane and collidine. These solvents may
be used singly or as mixtures in any proportion. Preferable
solvents are pyridine, dichloromethane and mixtures thereof.
Pyridine is most preferred. The reagent may be used at any
effective concentration for sulfurizing a phosphorous(III) linkage,
preferably between 0.01M to 1.5M, more preferably from 0.2 to 1.2M;
and most preferably from 0.5 to 1.0M.
The sulfurization reaction may be conducted at any convenient
temperature, preferably from 0 to 70.degree. C.; more preferably
from 10 to 40.degree. C.; and most preferably at about room
temperature, i.e., 18 to 25.degree. C. The sulfurization reaction
is preferably conducted for 30 seconds to 15 minutes, more
preferably, 1 to 15 minutes; and most preferably, 3 to 10 minutes.
Preferably, sulfurization is performed under anhydrous conditions
with the exclusion of air.
The present method for synthesizing a sulfurized oligonucleotide
analog may be applied to any oligonucleotide analog containing at
least one phosphorus(III) linkage which is amenable to
sulfurization. In particular, the present method is useful for
sulphurizing phosphite triesters, thiophosphite triesters, and
hydrogen phosphonates. More preferably, the phosphorus(III) linkage
is a phosphite triester or a thiophosphite triester. Most
preferably, the phosphorus(III) linkage is a phosphite
triester.
Detailed procedures for synthesizing oligonucleotide analogs
containing at least one phosphorus(III) linkages are well-known in
the art and are described in the following references: M. J. Gait
(ed.), Oligonucleotide Synthesis, A Practical Approach (ILR Press,
1984); J. S. Cohen (ed.), Oligonucleotides: Antisense Inhibitors of
Gene Expression, (CRC Press, Inc., Boca Raton, Fla., 1989).
Preferably, the dithiocarbonic acid diester polysulfide of the
present invention is used in conjunction with the phosphoramidite
or phosphorothioamidite synthetic approaches. Synthesis may be
conducted in solution phase or using solid phase techniques. More
preferably, the synthesis is conducted using a solid support. Most
preferably, the synthesis is conducted on a solid support using an
automated DNA synthesizer, e.g., an APPLIED BIOSYSTEMS model 380B
or a similar machine.
Preferably, this synthetic approach involves the following steps:
(1) deprotecting a blocked reactive functionality on the growing
oligonucleotide analog chain or on the first nucleoside analog
monomer, to produce a deblocked reactive functionality, (2)
reacting an appropriately blocked and protected nucleoside analog
phosphoramidite or phosphorothioamidite monomer with the deblocked
reactive functionality of the growing nucleotide analog chain,
preferably in the presence of an activator, to form an
oligonucleotide analog containing a phosphorus(III) linkage, (3)
capping any unreacted functionalities, and (4) sulfurizing the
newly-formed phosphorus(III) linkage with the thiodicarbonic acid
diester polysulfide to obtain the phosphorus atom in a sulfurized
pentacoordinate state.
The term "blocked" means that a reactive functionality, usually a
nucleophile, e.g., a 5' hydroxyl, is protected with a group that
may be selectively removed. Preferably, these blocking groups are
labile to dilute acid, e.g. dichloroacetic acid in dichloromethane,
and are stable to base. Preferable blocking groups include
4,4'-dimethoxytrityl (DmTr), monomethoxytrityl, diphenylmethyl,
phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthen-9-yl
(Mox). The 4,4'-dimethoxytrityl group is most preferred. Throughout
the present disclosure the labile 5' blocking group is represented
as "R.sup.6 ".
Any natural or non-natural heterocyclic base may be used in the
present invention, such as adenine, guanine, cytosine, thymine,
uracil, 2-aminopurine, inosine, substituted pyrimidines, e.g.,
5-methylcytosine, and 5-nitropyrrole. Other suitable heterocyclic
bases are described by Merigan et al., U.S. Pat. No. 3,687,808.
Preferably, the heterocyclic base is attached to C-1 of the sugar
moiety of nucleoside analog phosphoramidite (1) via a nitrogen of
the base. Throughout the present disclosure the heterocyclic base
is respresented as "B".
During synthesis these heterocyclic groups are preferably protected
to prevent any reactive group, e.g., an exocyclic amino group, to
prevent undesired side reactions. The term "protected" means that
reactive moieties such as exocyclic amino groups, 2'-hydroxyl
groups, oxygen or sulfur bonded to phosphorus atoms, and the like,
have protective groups which are generally removed after synthesis
of the oligonucleotide analog is completed. Preferably, these
protective groups are labile to a base and/or a nucleophile. This
term also includes oligonucleotide and nucleoside analogs which
have groups that do not require such protection, e.g., heterocyclic
bases such as thymine or abasic nucleosides.
Preferable protecting groups for the heterocyclic bases include
base labile groups. The exocyclic amino groups of the heterocyclic
groups are preferably protected with acyl groups that are removed
by base treatment after synthesis of the sulfurized oligonucleotide
analog. Preferably, these protecting groups are C.sub.2 -C.sub.10
acyl groups. N-benzoyl and N-isobutyryl protecting groups are
particularly preferred. Adenine is preferably protected as an
N.sup.2 -isobutyryl derivative. Guanine is preferably protected as
an N.sup.6 -isobutyryl derivative. Cytidine is preferably protected
as an N.sup.4 -benzoyl derivative.
The sulfurized nucleotide analogs of the present invention may be
substituted at the 2' position. Preferable 2' substituents are
groups that enhance the hybridization of an oligonucleotide analog
with its target nucleic acid, a group that improves the in vivo
stability of an oligonucleotide analog or enhances the
pharmacokinetic and/or pharamacodynamic properties of an
oligonucleotide analog. Examples of 2' substituents include
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl (such as CF.sub.3),
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, substituted O--C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, O--C.sub.6 -C.sub.14 aryl (such
as phenyl), substituted O--C.sub.6 -C.sub.14 aryl, O--C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
O--C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
O--C.sub.7 -C.sub.18 aralkyl (such as benzyl), substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl (such as
tert-butyldimethylsilyl), substituted O-tri-C.sub.1 -C.sub.8 -alkyl
silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1 -C.sub.8).sub.2,
NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1 -C.sub.8).sub.2 alkenyl,
S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1 -C.sub.8 alkenyl, NH.sub.2,
N.sub.3, NH--C.sub.1 -C.sub.8 -alkyl-NH.sub.2, polyalkylamino and
an RNA cleaving group. Preferable RNA cleaving groups include the
O-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl)-imidazol-4-yl)]}
group and the
O-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl-2-methoxy-5-acetylami
nomethyl)-imidazol-4-yl)]} group. These groups are discussed by
Cook et al, U.S. Pat. No. 5,359,051.
Preferably, the 2' substituents are hydrogen, hydroxyl, O--C.sub.1
-C.sub.8 alkyl, F, O--C.sub.1 -C.sub.8 -alkoxyamino and O--C.sub.1
-C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl. More preferably, the 2'
substituents are hydrogen, O--C.sub.1 -C.sub.8 alkyl, F and
O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl. Most
preferably, the 2' substituent is hydrogen or a methoxyethoxy
group. Throughout the present disclose, the 2' substituent is
respresented as "X".
A variety of protecting groups for the oxygen and sulfur atoms
attached to the phosphorus atom in the nucleoside analog
phosphoramidite and phosphorothioamidite, respectively, may be
used. These protecting groups are preferably removed at after
synthesis is complete. Preferably, these protecting groups are
labile to a base and/or a nucleophile. Most prefereably, these
protecting groups are removed by aqueous ammonium hydroxide.
Preferable protecting groups are 2-cyanoethyl, 4-cyano-2-butenyl,
2-diphenylmethylsilylethyl (DPSE) or a 2-N-amidoethyl group having
the formula R.sup.1 CONR.sup.2 CHR.sup.3 CHR.sup.4 --. These
protecting groups are preferably removed after synthesis,
preferably with an aqueous solution of ammonia at a temperature
between room temperature and 75.degree. C. In the present
invention, the terms "phosphodiester linkage", "phosphorothioate
linkage" and "phosphorodithioate linkage" describe these
internucleosidic linkages in protected or unprotected form.
Throughout the present disclosure, these phosphorous protecting
groups will be represented as "Pg". An oxygen atom or sulfur atom
attached to the phosphorous atom in a nucleoside analog
phosphoramidite or thiophosphoramidite or an oligonucleotide analog
is represenated as "Y". Preferably, Y is an oxygen atom.
The 4-cyano-2-butenyl protecting group is removed by
.delta.-elimination, preferably using the standard NH.sub.3
/H.sub.2 O deprotection conditions known in the art.
4-cyano-2-butenyl-protected nucleoside analog phosphoramidites may
be prepared with 4-cyano-2-butene-1-ol and appropriately protected
nucleoside analogs using known synthetic methodology. The synthesis
of 4-cyano-2-butene-1-ol is disclosed by Ravikumar et al, Synthetic
Communications 1996, 26(9), 1815-1819.
The 2-diphenylmethylsilylethyl (DPSE) protecting group is described
in Ravikumar et al., WO 95/04065. This protecting group may be
removed by treatment with a base, preferably aqueous ammonium
hydroxide. The DPSE group may also be removed with fluoride ion.
Preferably, the fluoride ion is provided from a salt such as a
tetraalkylammonium fluoride, e.g., tetrabutylammonium fluoride
(TBAF) or an inorganic fluoride salt, e.g., potassium fluoride or
cesium fluoride in a solvent such as tetrahydrofuran, acetonitrile,
dimethoxyethane or water.
The 2-N-amidoethyl group is described in the commonly assigned
application U.S. patent application Ser. No. 08/811232 (Title:
Protecting Group for Synthesizing Oligonucleotide Analogs, Attorney
Docket No. 7761-002-55). The 2-N-amidoethyl group has the formula
R.sup.1 CONR.sup.2 CHR.sup.3 CHR.sup.4 --, where R.sup.1 is C.sub.1
-C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms or substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms. More
preferably, R.sup.1 is C.sub.1 -C.sub.8 alkyl, substituted C.sub.1
-C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl,
substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl
containing up to three heteroatoms or substituted C.sub.3 -C.sub.11
hetaryl containing up to three heteroatoms. Even more preferably,
R.sup.1 is methyl, fluoromethyl, difluoromethyl or trifluoromethyl.
Most preferably, R.sup.1 is methyl, trifluoromethyl or phenyl.
The nitrogen atom of the 2-N-amidoethyl group may be unsubstituted,
i.e., R.sup.2 may be hydrogen, or substituted. Preferably, R.sup.2
is hydrogen, C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8
alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl,
substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl
containing up to three heteroatoms, substituted C.sub.3 -C.sub.11
hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18
aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms or
substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to
three heteroatoms. More preferably, R.sup.2 is hydrogen or C.sub.1
-C.sub.8 alkyl. Most preferably, R.sup.2 is hydrogen or methyl.
The ethyl moiety of the 2-N-amidoethyl group may be unsubstituted,
e.g., R.sup.3 and R.sup.4 may both be hydrogen. Alternatively, the
ethyl moiety may be substituted with groups that preferably do not
compromise the stability of the 2-N-amidoethyl group during
oligonucleotide analog synthesis and permit the protecting group to
be removed by treatment with a base and/or a nucleophile following
step-wise assembly of an oligonucleotide analog. Preferable R.sup.3
and R.sup.4 groups are hydrogen, C.sub.1 -C.sub.8 alkyl,
substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14
aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
substituted C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms or substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms. More
preferably, R.sup.3 is hydrogen or linear C.sub.1 -C.sub.8 alkyl.
Most preferably, R.sup.3 is hydrogen or methyl. More preferably,
R.sup.4 is hydrogen, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms or substituted C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms. Most preferably, R.sup.4 is hydrogen or
phenyl. The R.sup.3 and R.sup.4 groups are independently selected,
i.e., they may be the same or different.
Alternatively, R.sup.3 and R.sup.4, together with the carbon atoms
they are bonded to, may form a C.sub.3 -C.sub.8 cycloalkyl group, a
substituted C.sub.3 -C.sub.8 cycloalkyl group, a C.sub.2 -C.sub.8
heterocycloalkyl group containing up to three heteroatoms or a
substituted C.sub.2 -C.sub.8 heterocycloalkyl group containing up
to three heteroatoms. In this embodiment, R.sup.3 and R.sup.4,
together with the carbon atoms they are bonded to, preferably form
a C.sub.3 -C.sub.8 cycloalkyl group or a substituted C.sub.3
-C.sub.8 cycloalkyl group. More preferred cycloalkyl groups are
C.sub.4 -C.sub.7 or substituted C.sub.4 -C.sub.7 groups, with
C.sub.5 -C.sub.6 or substituted C.sub.5 -C.sub.6 groups most
preferred. An unsubstituted cycloalkyl group is particularly
preferred. An unsubstituted C.sub.6 cycloalkyl group is most
particularly preferred. The stereochemical relationship between the
N-amido group and Y may be cis or trans. A trans relationship is
preferred.
An allyl group is also a preferable protecting group for the oxygen
or sulfur atom attached to the phosphorus atom in an
oligonucleotide analog. The allyl protecting group is described in
U.S. Pat. No. 5,026,838. The term "allyl group" includes allyl,
methallyl, crotyl, prenyl, geranyl, cinnamyl and p-chlorocinnamyl
groups. The number of carbon atoms in these groups is preferably 3
to 10. Preferably, the allyl group is an unsubstituted allyl group.
The allyl group may be removed with a palladium(O) compound and a
nucleophilic agent, such as an amine or a formic acid salt, under
neutral conditions at room temperature. A preferred reagent is
tetrakis(triphenylphosphine) palladium(O) and n-butylamine in
tetrahydrofuran.
An activator is generally used in the coupling of a deblocked
reactive functionality on the oligonucleotide or nucleoside analog
and the phosphoramidite or phosphorothioamidite monomer. Preferable
activators are well-known in the art, such as 1H-tetrazole,
5-(4-nitrophenyl)-1H-tetrazole and diisopropylamino tetrazolide.
1H-Tetrazole is most preferred.
A capping step is preferably used after this coupling reaction to
permanently block all uncoupled reactive functionalities. Suitable
capping reagents are well-known in the art. A preferable capping
reagent is acetic anhydride/lutidine/THF (1:1:8) with
N-methylimidazole/THF.
When synthesis is performed by solution phase methods, the 3'
terminal hydroxyl group of an oligonucleotide analog is preferably
protected to prevent the 3' hydroxyl group from participating in
any undesired side reactions. Preferably, the terminal 3' hydroxyl
group is protected with a group which may be removed selectively
without removing any other protecting groups. A C.sub.2 -C.sub.10
acyl group is preferred. The acetyl or levulinyl group is more
preferred. The levulinyl group is most preferred. Throughout the
present disclosure this 3' protecting group is represented as
"R.sup.7 ".
The sequential addition of nucleoside analog phosphoramidites or
phosphorothioamidites may be repeated until an oligonucleotide
analog having the desired sequence length is obtained. The length
of the sulfurized oligonucleotide analog is preferably 2 to 200
monomer units; more preferably, 2 to 100 monomer units; even more
preferably, 2 to 50 monomer units; and, most preferably, 2 to 25
monomer units. These ranges include all subranges therebetween.
In a preferred embodiment of the present invention, a sulfurized
oligonucleotide analog is synthesized on a solid support. Suitable
solid supports include controlled pore glass (CPG);
oxalyl-controlled pore glass, see for example Alul et al, Nucleic
Acids Research 1991, 19, 1527; TENTAGEL Support, see Wright et al,
Tetrahedron Letters 1993, 34, 3373; POROS, a polystyrene resin
available from PERCEPTIVE BIOSYSTEMS; and a
polystyrene/divinylbenzene copolymer. Controlled pore glass is the
most preferred solid support. Throughout the present discloure the
solid support is represented as "S.sup.p ".
The oligonucleotide analog is preferably attached to the solid
support by a group which may be easily cleaved to release the
oligonucleotide analog from the solid support when synthesis is
complete. Preferably, this group may be cleaved upon exposure to a
base and/or a nucleophile. More preferably, the group is an acyl.
Most preferably, the group is a carboxyl group esterifed with the
terminal 3' hydroxyl group of the oligonucleotide analog. The group
linking an oligonucleotide analog to a solid support is represented
as "L" in the present disclosure.
The present invention includes sulfurized oligonucleotide analogs
containing phosphorothioate, phosphorodithioate, and phosphodiester
linkages in any combination. The sulfurized oligonucleotide analogs
of the present invention may contain only sulfurized linkages,
e.g., phosphorothioate and/or phosphorodithioate. The
oligonucleotide analogs may also contain one or more phosphodiester
linkages in addition to the sulfurized linkages. In a preferred
embodiment, the sulfurized oligonucleotide analog contains both
phosphorothioate and phosphodiester linkages. In another preferred
embodiment, the oligonucleotide contains both phosphorodithioate
and phosphodiester linkages.
Phosphodiester linkages are formed by oxidizing a phosphorous(III)
linkage with any suitable oxidizing reagent known in the art, e.g.,
I.sub.2 /THF/H.sub.2 O, H.sub.2 O.sub.2 /H.sub.2 O, tert-butyl
hydroperoxide or a peracid, such as m-chloroperbenzoic acid.
I.sub.2 /THF/H.sub.2 O is a preferred oxidizing agent.
In a preferred embodiment, the oligonucleotide analog containing at
least one phosphorous(III) linkage has the formula: ##STR4## where
each Pg is independently a group labile to a base and/or a
nucleophile or an allyl group;
R.sup.6 is a labile blocking group;
each B is independently an unprotected or protected heterocyclic
base;
each X is independently selected from the group consisting of
hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted
C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl
containing up to three heteroatoms, substituted C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, C.sub.6
-C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3
-C.sub.11 hetaryl containing up to three heteroatoms, substituted
C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms,
C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl,
C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl
containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl,
substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8
heterocycloalkyl containing up to three heteroatoms, substituted
O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three
heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6
-C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up
to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted
O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms, substituted
O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three
heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl,
O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino,
O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1
-C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1
-C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1
-C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1
-C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8
-alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group;
each Y is independently O or S;
each Z is independently O or S;
L is a group labile to a nucleophile and/or base;
S.sup.p is a solid support; and
m is 0 or a positive integer.
Preferred protecting groups, Pg, include 2-cyanoethyl,
4-cyano-2-butenyl, 2-diphenylmethylsilylethyl (DPSE) and a
2-N-amidoethyl group.
In a preferred embodiment, the oligonucleotide analog containing a
phosphorous(III) linkage contains 2 to 100 monomer units, i.e., m
is 0 to 98; more preferably, from 2 to 50 monomer units, i.e., m is
0 to 48; and most preferably from 2 to 25 monomer units, i.e., m is
0 to 23.
Treating the above oligonucleotide analog with a dithiocarbonic
acid diester polysulfide affords the corresponding sulfurized
oligonucleotide analog having the formula: ##STR5## where Pg,
R.sup.6, B, X, Y, Z, L, S and m are defined above. Z is an oxygen
atom or a sulfur atom. Appropriate choice of Z allows the
internucleosidic linkage containing Z to be a phosphodiester,
phosphorothioate or phosphorodithioate linkage, depending on
selection of Y. For example, when Y and Z are both oxygen the
linkage is a phosphodiester. When Y is oxygen and Z is sulfur, or
vice versa, the linkage is a phosphorothioate. When Y and Z are
both sulfur the linkage is a phosphorodithioate. Preferably, Y is
an oxygen atom and Z is a sulfur atom which has been introduced
using the dithiocarbonic acid diester polysulfide described above.
It is to understood that the terms phosphodiester, phosphorothioate
and phosphorodithioate refer to the internucleotide linkage after
removal of the Pg groups.
Following sulfurization, the sequence of the sulfurized
oligonucleotide analog may be further extended. Alternatively, the
sulfurized oligonucleotide analog may be deprotected and removed
from the solid support to afford the corresponding deprotected
sulfurized analog. The deprotected sulfurized analog may contain
phosphorothioate, phosphorodithioate and phosphodiester linkages,
in any combination.
In another preferred embodiment of the present invention, the
oligonucleotide analog containing at least one phosphorous(III)
linkage is a dinucleotide having the formula: ##STR6## where
R.sup.7 is a group labile to a base and/or a nucleophile.
Sulfurization of the dinucleotide analog with the dithiocarbonic
acid diester polysulfide affords the corresponding sulfurized
dinucleotide analog having the formula: ##STR7##
The present invention also provides a method for synthesizing a
sulfurized oligonucleotide analog by:
(a) providing a nucleoside analog having a blocked hydroxyl
group;
(b) deblocking the blocked hydroxyl group to obtain a free hydroxyl
group;
(c) reacting the free hydroxyl group with a protected nucleoside
analog phosphoramidite having a blocked hydroxyl group to produce
an oligonucleotide analog containing a phosphorous(III) linkage and
a blocked hydroxyl group;
(d) reacting the phosphorous(III) linkage with a reagent selected
from the group consisting of an oxidizing agent and a
dithiocarbonic acid diester polysulfide having the formula:
##STR8## to produce an oxidized or sulfurized phosphorous(V)
linkage; (e) repeating steps (b) through (d) at least once to
produce a sulfurized oligonucleotide analog; where the
oligonucleotide analog contains at least one sulfurized
phosphorous(V) linkage. Preferably, each phosphorous(V) linkage is
sulfurized.
A preferred nucleoside analog phosphoramidite having a blocked
hydroxyl group used in step (c) has the formula: ##STR9##
The R.sup.5 groups are chosen such that the nucleoside analog
phosphoramidite preferably couples efficiently with a reactive
group on the on the growing oligonucleotide analog chain, e.g., a
5' hydroxyl group, to form a phosphorous(III) internucleotide
linkage. The R.sup.5 groups are independently selected, i.e., they
may be the same or different. Preferable R.sup.5 groups are C.sub.1
-C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2
-C.sub.8 heterocycloalkyl containing up to three heteroatoms,
substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to
three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6
-C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three
heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to
three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7
-C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing
up to three heteroatoms or substituted C.sub.4 -C.sub.15
heterocycloaralkyl containing up to three heteroatoms; or both
R.sup.5 groups together with the nitrogen atom they are bonded to
form a C.sub.2 -C.sub.8 heterocycloalkyl group containing up to
three heteroatoms, a substituted a C.sub.2 -C.sub.8
heterocycloalkyl group containing up to three heteroatoms, a
C.sub.3 -C.sub.11 hetaryl group containing up to three heteroatoms
or a substituted C.sub.3 -C.sub.11 hetaryl group containing up to
three heteroatoms. More preferably, the R.sup.5 groups are each a
C.sub.1 -C.sub.8 alkyl group or together with the nitrogen atom
they are bonded to form a C.sub.2 -C.sub.8 heterocycloalkyl group
containing up to three heteroatoms. Even more preferably, each
R.sup.5 group is a branched C.sub.1 -C.sub.8 alkyl group. Most
preferably, both R.sup.5 groups are isopropyl.
In a preferred embodiment, the nucleoside analog in step (a) is
attached to a solid support. A preferred nucleoside analog attached
to a solid support has the formula: ##STR10##
In another embodiment, the oligonucleotide analog containing a
phosphorous(III) linkage and a blocked hydroxyl group is attached
to a solid support and has the formula: ##STR11## and the resulting
sulfurized oligonucleotide analog has the formula: ##STR12##
The sulfurized oligonucleotide analog synthesized on a solid
support may be removed from the support, preferably by base
treatment. Preferably, all of the protecting groups are removed
during cleavage from the solid support. Preferable reagents for
cleaving the sulfurized oligonucleotide analog from the solid
support are aqueous ammonium hydroxide and ammonia/methanol
solutions. The simultaneous deprotection and removal of the
sulfurized oligonucleotide analog is preferably accomplished in
aqueous ammonium hydroxide at a temperature between room
temperature, i.e., 18 to 25.degree. C., and 75.degree. C.; more
preferably, between room temperature and 65.degree. C.; and most
preferably, between room temperature and 60.degree. C. A
temperature of 55.degree. C. is particularly preferred. The
deprotection reaction time is preferably 1 to 30 hours; more
preferably, 1 to 24 hours; and most preferably, 12-24 hours. The
concentration of ammonium hydroxide in the solution used for
deprotection is preferably 20 to 30% by weight; more preferably, 25
to 30% by weight; and most preferably, 28 to 30% by weight.
In a preferred embodiment, the sulfurized oligonucleotide analog
released from the solid support has the formula: ##STR13## where
each B is preferably an unprotecteted heterocyclic base.
Each internucleotide linkage of the sulfurized oligonucleotide
analog released from the solid support may be ionized, depending on
the pH, temperature and salt conditions. Each internucleotide
linkage will be ionized in aqueous solution at physiologic pH,
temperature and salt conditions, i.e., pH 7.2, 37.degree. C. and
about 150 mM monovalent salts.
Having generally described this invention, a further understanding
can be obtained by reference to certain specific examples which are
provided herein for purposes of illustration only and are not
intended to be limiting unless otherwise specified.
EXAMPLES
Example 1
Diethyldithiocarbonate Disulfide
Potassium ethyl xanthate (120 g) was dissolved in a minimum amount
of water. Iodine was added to this solution portionwise at
10.degree. C. until a dark brown color persisted. A small amount of
aqueous saturated Na.sub.2 S.sub.2 O.sub.3 was added to quench the
reaction and remove the dark brown color from the solution. The
resulting solution was extracted with ether. The combined ether
layers were washed three times with water, dried and concentrated
to afford diethyldithiocarbonate disulfide as a pale yellow
solid.
Example 2
Synthesis of a T-T Phosphorothioate Dimer
100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine attached
to a controlled pore glass (CPG) support by an ester linkage was
added to a glass reactor, and solution of 2% dichloroacetic acid in
dichloromethane (volume/volume) was added to deprotect the
5'-hydroxyl group. The product was first washed with
dichloromethane and then with acetonitrile. A 0.2M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution
of 1H-tetrazole in acetonitrile were added, and allowed to react at
room temperature for 5 minutes. The product was first washed with
acetonitrile, and then a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added and allowed reacted at room
temperature for 100 seconds. This sulfurization step was repeated
for 100 seconds. The CPG was washed with acetonitrile and then a
solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methylimidazole/THF was added to cap unreacted 5'-hydroxyl
groups. The CPG was then washed with acetonitrile. The CPG was
treated with 30% aqueous ammonium hydroxide solution for 90
minutes. The aqueous solution was filtered, concentrated under
reduced pressure to afford the desired T-T phosphorothioate
dimer.
Example 3
Synthesis of a C-T Phosphorothioate Dimer
100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine attached
to a CPG support by an ester linkage was added to a glass reactor,
and a solution of 2% dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group. The
CPG was then washed with acetonitrile. A 0.2M solution of N.sup.4
-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution
of 1H-tetrazole in acetonitrile was added, and allowed to stand at
room temperature for 5 minutes. The CPG was then washed with
acetonitrile, followed by addition of a 1M solution of
diethyldithiocarbonate disulfide in pyridine. The sulfurization
reaction was allowed to procede at room temperature for 100
seconds. The sulfurization step was repeated for an additional 100
seconds. The support was then washed with acetonitrile followed by
addition of a solution of acetic anhydride/lutidine/THF (1:1:8),
and N-methylimidazole/THF to cap unreacted 5'-hydroxyl groups.
After capping, the CPG was washed with acetonitrile. The CPG was
treated with 30% aqueous ammonium hydroxide solution for 90 minutes
and then incubated at 55.degree. C. for 12 hours. The aqueous
solution was filtered, concentrated under reduced pressure to
afford the desired C-T phosphorothioate dimer.
Example 4
Synthesis of G-T Phosphorothioate Dimer
100 milligrams (4 mmole) of 5'-O-dimethoxytritylthymidine attatched
to a CPG support by an ester linkage was added to a glass reactor,
and a 2% solution of dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group. The
CPG was washed with dichloromethane and then washed with
acetonitrile. A 0.2M solution of N.sup.2
-isobutyrl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-cyanoethy
l N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M
solution of 1H-tetrazole in acetonitrile was added, and reacted at
room temperature for 5 minutes. The product was washed with
acetonitrile, and then a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added. The sulurization reaction was
allowed to proceed at room temperature for 100 seconds. The
sulfurization step was repeated for an additional 100 seconds. The
support was washed with acetonitrile and then a solution of acetic
anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added
to cap any unreacted 5'-hydroxyl groups. The CPG was then washed
with acetonitrile. The CPG was treated with 30% aqueous ammonium
hydroxide solution for 90 minutes at room temperature and then
incubated at 55.degree. C. for 1 hour. The aqueous solution was
filtered and concentrated under reduced pressure to give the
desired T-T phosphorothioate dimer.
Example 5
Synthesis of a 5'-TTTTTTT-3' Phosphorothioate Heptamer
50 milligrams (2 mmole) of 5'-O-dimethoxytritylthymidine attached
to a CPG support by an ester linkage was added to a glass reactor,
and a 2% solution of dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group. The
CPG was then washed with acetonitrile. A 0.2M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution
of 1H-tetrazole in acetonitrile was added, and allowed to react at
room temperature for 5 minutes. The CPG was washed with
acetonitrile, and then a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added and allowed to react at room
temperature for 100 seconds. This sulfurization step was repeated
for 100 seconds. The support was washed with acetonitrile, and then
a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methylimidazole/THF was added to cap any unreacted 5'-hydroxyl
groups. After capping, the solid support was washed with
acetonitrile. This complete cycle was repeated five times to afford
the protected thymidine heptamer. The support containing the
compound was treated with 30% aqueous ammonium hydroxide solution
for 90 minutes at room temperature. The aqueous solution is
filtered, and concentrated under reduced pressure to afford the
desired phosphorothioate heptamer 5'-TTTTTTT-3'.
Example 6
Synthesis of 5'-d(GACTT)-3' Phosphorothioate Pentamer
50 milligrams (2 mmole) of 5'-O-dimethoxytritylthymidine bound to a
CPG controlled pore glass support through an ester linkage was
added to a glass reactor, and a 2% solution of dichloroacetic acid
in dichloromethane (volume/volume) was added to deprotect the
5'-hydroxyl group. The CPG was then washed with acetonitrile. A
0.2M solution of
5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution
of 1H-tetrazole in acetonitrile were added, and allowed to react at
room temperature for 5 minutes. The CPG was washed with
acetonitrile, and then a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added and allowed to react at room
temperature for 100 seconds. This sulfurization step was repeated
for 100 seconds. The support was washed with acetonitrile and then
a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methylimidazole/THF was added to cap the unreacted 5'-hydroxyl
groups. After capping, the support was washed with
acetonitrile.
A solution of 2% dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group,
followed by washing with acetonitrile. A 0.2M solution of N.sup.4
-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution
of 1H-tetrazole in acetonitrile were added, and allowed to react at
room temperature for 5 minutes. After washing the solid support
with acetonitrile, a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added and allowed to react at room
temperature for 100 seconds. This sulfurization step was repeated
for 100 seconds. The support was washed with acetonitrile and then
a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methylimidazole/THF was added to cap any unreacted 5'-hydroxyl
groups. The support was washed with acetonitrile.
A solution of 2% dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group. The
CPG was washed with acetonitrile. A 0.2M solution of N.sup.6
-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(2-cyanoethyl
N,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a
0.4M solution of 1H-tetrazole in acetonitrile were added and
allowed to react at room temperature for 5 minutes. The product was
washed with acetonitrile, and then a 1M solution of
diethyldithiocarbonate disulfide in pyridine was added and allowed
to react at room temperature for 5 minutes. This sulfurization step
was repeated for 5 minutes. The support was washed with
acetonitrile and then a solution of acetic anhydride/lutidine/THF
(1:1:8), and N-methylimidazole/THF was added to cap the unreacted
5'-hydroxyl groups. The product is washed with acetonitrile.
A solution of 2% dichloroacetic acid in dichloromethane
(volume/volume) was added to deprotect the 5'-hydroxyl group. The
product was washed with acetonitrile. Then, a 0.2M solution of
N.sup.2
-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-cyanoeth
yl N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M
solution of 1H-tetrazole in acetonitrile were added, and allowed to
react at room temperature for 5 minutes. The product was washed
with acetonitrile, and then a 1M solution of diethyldithiocarbonate
disulfide in pyridine was added and allowed to react at room
temperature for 100 seconds. This sulfurization step was repeated
for 100 seconds. The support was washed with acetonitrile and then
a solution of acetic anhydride/lutidine/THF (1:1:8), and
N-methylimidazole/THF was added to cap any unreacted 5'-hydroxyl
groups. The product was washed with acetonitrile.
The CPG was treated with 30% aqueous ammonium hydroxide solution
for 90 minutes at room temperature and then incubated at 55.degree.
C. for 24 hours. The aqueous solution was filtered, concentrated
under reduced pressure to give the desired 5'-d(GACTT)-3'
phosphorothioate tetramer.
Example 7
Synthesis of Homo-Thymidine Phosphorothioate 19-mer
A 19-base homo-thymidine phosphorothioate oligonucleotide was
synthesized by the phosphoramidite method on an automated
synthesizer (ABI model 39OZ, Foster City, Calif.). The standard
synthesis protocol was followed, except that in place of the
oxidation step a sulfurization step was substituted, and this step
preceded the capping step. In other words, synthesis consisted of
repeated cycles of detritylation, coupling, sulfurization, and
capping. Separation of the final product from the synthesis column
and purification were accomplished using well-known methods. The
sulfurization step involved exposing the growing chain to a 1M
solution of diethyldithiocarbonate disulfide in pyridine for 100
seconds at room temperature.
The yield of trityl cation released during the detritylation steps
averaged 99%. The trityl yield is both a measure of coupling
efficiency and a measure of the extent of sulfurization, since
non-sulfurized trivalent phosphorus linkages in the oligonucleotide
are labile to cleavage during detritylation. The 19-mer was cleaved
from the support and deprotected with concentrated ammonium
hydroxide under standard conditions and isolated using techniques
well-known in the art.
Example 8
Large-Scale Synthesis of a 20-mer Phosphorothioate Oligonucleotide
Analog
A 20-base phosphorothioate oligonucleotide analog of sequence
5'-TCCCGCCTGTGACATGCATT-3' was synthesized by the phosphormidite
method on an OligoPilot automated DNA synthesizer (available from
Pharmacia, Sweden). The standard synthesis protocol was used with
the oxidation step replaced with a sulfurization step with
diethyldithiocarbonate disulfide. Sulfurization of each
phosphorous(III) linkage was accomplished by exposing the
oligonucleotide analog to a 0.2M solution of diethyldithiocarbonate
disulfide in pyridine/dichloromethane (1:1 v/v) for 100 seconds at
room temperature. The resulting 20-mer phosphorothioate
oligonucleotide analog was deprotected and cleavage from the solid
support with aqueous ammonium hydroxide and purified by well-known
methods.
All references cited in the present application are hereby
incorporated by reference in their entirety.
Obviously, numerous modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *